Antigen Immobilization
It is a general principle of solid phase immuncassays that most water soluble proteins will bind irreversibly to certain types of plastic, particularly specially formulated polystyrene. However, the protein binding capacity of the plastic is very small. The purity of a protein and its absolute concentration in a mixture of proteins will determine if that protein will bind to a plastic surface in sufficient quantity to be useful as a solid phase antigen. Therefore, many proteins must be purified and concentrated prior to binding to a plastic surface for use as antigens in a solid phase immunoassay.
A plastic surface may first be coated with an antibody or other protein to enhance binding of the antigen of interest. (Moore et al, AIDS, 4:155-163, 1989.) In this type of "sandwich" immunoassay, an antibody is first passively coated to a plastic surface and then reacted with a solution containing the antigen of choice. Antigen thus immobilized is used in an immunoassay to detect antibodies in sera or other body fluids and tissue culture fluids. The immobilizing or "capture" antibody should not bind to the same antigenic sites to which the test antibodies are expected to bind.
Another coating substance by which antigens may be immobilized on plastic surfaces is Poly-L-Lysine (PLL). However, PLL, like plastic alone, is not able to select the type of proteins that are immobilized. Thus, prior to using PLL, the antigen of choice must be highly purified.
Solid Phase Immunoassays to Detect Antibodies to Viral Antigens
Today's research scientists and physicians require rapid, accurate and inexpensive methods to detect antibodies to a variety of viruses. In order to prepare assay kits for the marketplace, it is necessary to produce sufficient quantities of viral antigens. However, relatively small amounts of virus (particularly human retroviruses) are released in cultures of cells infected with the viruses. To obtain the quantities of virus antigens required to prepare large numbers of passively coated plastic surfaces, it is necessary to grow very large quantities of virus infected cells in tissue culture and then to separate the virus particles from the other protein constituents of the culture medium, especially the protein-rich serum used in the medium.
An additional consideration is the protein distribution of the virus particles. For example, The predominant protein species of retroviruses such as HIV-1 are found in the viral core, however the relatively minor glycoproteins found in the viral envelope are much more immunogenic. (Kalyanaraman et al, AIDS Research & Human Retroviruses, 4:319-329, 1988.) During the procedures of purification and inactivation of human retroviruses, a large proportion of the highly aritigenic envelope glycoprotein component is lost. (Moore et al, AIDS, 3:155-163, 1989.) Therefore, coating procedures which utilize purified retrovirus (such as HIV-1) provide relatively more core antigens than envelope antigens for immobilization. Moreover, no process to date has been able to completely purify virus from non-viral cellular proteins. Contaminating proteins, including histocompatibility antigens, may be co-purified with the virus and may passively bind to plastic surfaces. Such contaminants account for some of the false positive reactions obtained in serologic testing using these methods.
Because in general only small quantities retroviruses are released into the culture medium by infected cells, large amounts of virus producer cells must be grown in order to produce commercially useful quantities of concentrated, purified virus needed for making passively coated, solid phase immunoassays. For example, as much as 10-100 liters of virus-producing cells infected with human retroviruses such as HIV-1 or HTLV-I are typically needed to produce commercially useful quantities of antigens. Producing and processing such volumes of these viruses is both expensive and potentially biohazardous.
Recombinant DNA techniques may also be used to produce large amounts of virus encoded antigens. However, these antigens must still be purified from their producing cells before they can be passively absorbed to plastic surfaces creating similar problems as discussed above. The production of recombinant proteins is also expensive.
The available methods for producing viral antigens needed for solid phase immunoassays to detect antibodies against viruses such as HIV-1, HIV-2 and HTLV-1 require costly materials, costly processing, and considerable biohazards. The antigenic end-product provided by these methods is an antigen preparation containing a disproportionate amount of core antigens relative to the more desirable envelope glycoprotein antigens. In addition, the direct coating technique offers little control over which viral proteins become immobilized, and thus over the nature of the immunoassay.
It would be of great utility to provide a method to selectively immobilize viral glycoproteins which does not require that the virus be purified before coating the solid phase; which does not require large quantities of virus-producing cultures; which is inexpensive; and which provides immobilized viral antigen, for solid phase immunoassays that perform as well as or better than existing methods; and which requires no special procedures or equipment.